Post-crotonylation oxidation by a monooxygenase promotes acetyl-CoA synthetase degradation in Streptomyces roseosporus

Protein post-translational modifications (PTMs) with various acyl groups play central roles in Streptomyces. But whether these acyl groups can be further modified, and the influences of these potential modifications on bacterial physiology have not been addressed. Here in Streptomyces roseosporus with rich crotonylation, a luciferase monooxygenase LimB is identified to elaborately regulate the crotonylation level, morphological development and antibiotic production by oxidation on the crotonyl groups of an acetyl-CoA synthetase Acs. This chemical modification on crotonylation leads to Acs degradation via the protease ClpP1/2 pathway and lowered intracellular crotonyl-CoA pool. Thus, we show that acyl groups after PTMs can be further modified, herein named post-PTM modification (PPM), and LimB is a PTM modifier to control the substrate protein turnover for cell development of Streptomyces. These findings expand our understanding of the complexity of chemical modifications on proteins for physiological regulation, and also suggest that PPM would be widespread.


Figure
Figure S1 LimB homologs regulates crotonylation.a.The epoxidation reaction catalyzed by MsnO8 for the biosynthesis of mensacarcin.b.Domain organization of MsnO8 and LimB.The Flavin_utilizing_monooxygenase superfamily domain was shown in both proteins.c. S. roseosporus LimB (Orf6299) and its homologs from S. coelicolor M145 (Q9X888), S. albus J1074 (WP_0155508125.1)and E. coli DH5 (WP_000130380.1) were expressed in S. roseosporus L30 under the promoter ermEp* and tagged with 3FLAG.The cell lysate of WT and recombinant strains was prepared and subject for Western blot with -Kcr or -FLAG antibody, and the total protein was stained with Coomassie blue for the loading control.

Figure
Figure S2 Dry weight of wild type (WT), the limB null mutant (limB) and limB overexpression strain (WT + ermEp*-limB) in the YEME culture.Data are mean ± SEM for n = 3 biologically independent samples.

Figure
Figure S5 Proteasome is required for LimB degradation.3FLAG-tagged LimB was expressed in WT and the prcB/A mutant, respectively, and the total cell lysate was prepared and subject to Western blot with an -FLAG antibody or Coomassie blue staining as the loading control.

Figure
Figure S7 MS/MS analysis of crotonylation on Acs K116.Crotonylation was calculated based on the molecular weight difference values between y6 and y5 (801.41 -605.29 = 196.12).

Figure
Figure S8 MS/MS analysis of crotonylation on Acs K133.Crotonylation was calculated based on the molecular weight difference values between y4 and y3 (543.29 -347.17= 196.12).

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Figure S9 MS/MS analysis of crotonylation on Acs K367.Crotonylation was calculated based on the molecular weight difference values between y9 and y8 (1097.56-901.44 = 196.12).

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Figure S10 MS/MS analysis of crotonylation on Acs K496.Crotonylation was calculated based on the molecular weight difference values between y9 and y8 (1024.51-828.39 = 196.12).

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Figure S11 MS/MS analysis of crotonylation on Acs K593.Crotonylation was calculated based on the molecular weight difference values between y3 and y2 (440.29 -244.17= 196.12).

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Figure S13 Bacterial two-hybrid system was used to screen interacting proteins.The acs gene was cloned into pKT25, and clpP1, clpP2, clp A, clpB, lon, ftsH, clpX, prcA, prcB, pup, orf967, orf3399, orf4198, orf4819 and orf6758 were cloned into pUT18.The empty plasmids were used as a negative control.The zip plasmids were used as a positive control.

Figure
Figure S14 In-frame deletion of acs in S. roseosporus L30.a. Schematic diagram of acs knock-out.The expected DNA fragment sizes were shown.b.Confirmative PCR for acs deletion.The fragments of 3.2 kb and 1.2 kb were amplified from the genomic DNA of wild type and the Δacs mutant, respectively.

Figure
Figure S15 In-frame deletion of limB in S. roseosporus L30.a. Schematic diagram of limB knock-out.The expected DNA fragment sizes were shown.b.Confirmative PCR for limB deletion.The fragments of 1.8 kb and 1.0 kb were amplified from the genomic DNA of wild type and the ΔlimB mutant, respectively.

Figure
Figure S16 In-frame deletion of clpP2 in S. roseosporus L30.a. Schematic diagram of clpP2 knock-out.The expected DNA fragment sizes were shown.b.Confirmative PCR for clpP2 deletion.The fragments of 1.6 kb and 1.0 kb were amplified from the genomic DNA of wild type and the ΔclpP2 mutant, respectively.

Table S1
Putative luciferase monooxygenases from S. roseosporus through Local BLAST using the MsnO8 protein sequence, which corresponds to S. griseus.TableS2The acyl-and acetyl-CoA synthetase libraries and crotonyl lysine modification sites in this study.Identification and analysis of crotonylated proteins were provided by PTM Biolabs Inc (Hangzhou, China) 1 .

Table S3 Putative
Clp proteases of S. roseosporus L30 through Local Blast.
roseosporusThis studyΔlimBIn-frame deletion of limb in S. roseosporus This study Δacs In-frame deletion of acs in S. roseosporus This study WT + ermEp*-limB limB overexpressed under ermEp* in S. roseosporus This study WT + ermEp*-acs acs overexpressed under ermEp* in S.